CB 2 receptor antibody signal specificity: correlations with the use of partial CB 2 -knockout mice and anti-rat CB 2 receptor antibodies

Hai ying Zhang, Hui Shen, Chloe J. Jordan, Qing rong Liu, Eliot L. Gardner, Antonello Bonci, Zheng xiong Xi

Research output: Contribution to journalArticle

Abstract

Cannabinoid CB 1 receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB 2 receptors (CB 2 Rs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CB 2 Rs in the brain. However, this finding is disputed because of the specificity of CB 2 R antibody signals. We used two strains of currently available partial CB 2 -knockout (CB 2 -KO) mice as controls, four anti-rat or anti-mouse CB 2 R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB 2 R-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB 2 -like immunostaining in mouse midbrain dopamine neurons. CB 2 R deletion in CB 2 -KO mice may reduce or leave CB 2 R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB 2 R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB 2 -KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB 2 -KO mice. Quantitative RT-PCR assays detected CB 2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB 2 -KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB 2 R-specific. Non-specific binding may be related to the expression of mutant or truncated CB 2 R-like proteins in partial CB 2 -KO mice and the use of anti-rat CB 2 antibodies because the epitopes are different between rat and mouse CB 2 Rs.

Original languageEnglish (US)
Pages (from-to)398-409
Number of pages12
JournalActa Pharmacologica Sinica
Volume40
Issue number3
DOIs
StatePublished - Mar 1 2019

Keywords

  • CB -KO mice
  • CB gene
  • CB receptor antibody
  • CB receptors
  • cannabinoid
  • midbrain dopamine neurons
  • specific immune peptides
  • specificity
  • spleen

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)

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